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Mitochondrial Ca2+ and ROS take center stage to orchestrate TNF-mediated inflammatory responses
Laura A. Dada and Jacob I. Sznajder
Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Proinflammatory stimuli induce inflammation that may progress to sepsis or chronic inflammatory disease. The cytokine TNF- is an important endotoxin-induced inflammatory glycoprotein produced predominantly by macrophages and lymphocytes. TNF- plays a major role in initiating signaling pathways and pathophysiological responses after engaging TNF receptors. In this issue of JCI, Rowlands et al. demonstrate that in lung microvessels, soluble TNF- (sTNF-) promotes the shedding of the TNF- receptor 1 ectodomain via increased mitochondrial Ca2+ that leads to release of mitochondrial ROS. Shedding mediated by TNF-converting enzyme (TACE) results in an unattached TNF receptor, which participates in the scavenging of sTNF-, thus limiting the propagation of the inflammatory response. These findings suggest that mitochondrial Ca2+, ROS, and TACE might be therapeutically targeted for treating pulmonary endothelial inflammation.
Role of TNF- in sepsis Duringsepsis,activationofproinflammatory pathways leads to dysfunction of mitochondria and cells, which contributestomultiorganfailureandpoor outcomes. TNF- simultaneously activatestheJNKandNF-kBpathwaysand increases expression of the mitochondrialantioxidantproteinSOD2,leading toanincreaseinH2O2andtheinactivation of JNK phosphatases. TNF receptorsaredifferentiallyexpressedinvariouscellsandtissuesandplayakeyrole in modulating the cellular inflammatoryresponses(1).Theseverityofinjury to the pulmonary endothelium during sepsisisdeterminedbyacomplexinterplayamongproinflammatorycytokines
Conflict of interest:J.I.Sznajderistheeditorofthe American Journal of Respiratory and Critical Care Medicine andreceivesastipendfromtheAmericanThoracicSocietyforthisfunction. Citation for this article: J Clin Invest.doi:10.1172/ JCI57748.

TACE-mediatedsheddingoftheTNFR1 ectodomain,whichinturnmodulatedthe endothelialinflammatoryresponse. Mitochondrial function and Ca2+ signaling Ca 2+ uptake by mitochondria is highly regulated,andthelevelsofmitochondrial Ca2+playacentralroleinthemetabolic (ATP production) and signaling (ROS production,releaseofproapoptoticmolecules)functionsoftheorganelle(Figure 1A).Inpatientswithacutesepsis,mitochondrial function is impaired (Figure 1B), and in patients with septic shock, theseverityofimpairmentinmitochondrialfunctionisassociatedwithadverse clinicaloutcomes(6,7).Normalization ofmitochondrialbiogenesisisnecessary torestoreoxidativemetabolismduring sepsis (Figure 1C) so that adequate O 2 deliveryandO2tissueconsumptioncan resume,allowingcellularmetabolicneeds tobemet(8,9). Rowlandsetal.suggestthattheTNFmediatedincreaseofmitochondrialCa2+ isdependentonERCa2+releaseleading toIP3-mediateddepletionofintracellular stores(5).Itisknownthatstoredepletiontriggerstheactivationofaninward rectifyingCa 2+currentmediatedeither bytheactionofspecializedCa2+release activated (CRAC) channels or by the openingofvoltage,receptor,orsecond messenger operated channels. TNF- inducedincreasesincytosolicCa2+lead toincreasedmitochondrialCa2+uptake through mitochondrial voltage-dependentanionchannels,themitochondrial

(includingTNF-),adhesionmolecules expressed on the endothelial cells, and leukocytesrecruitedtothesiteofinjury. Overwhelmingsepsisorperturbationof theadaptiveresponsesmayleadtoexacerbationofinflammation(2).Animportant mechanism to limit the TNF-mediated proinflammatoryresponseinvolvessheddingoftheTNF-receptor1(TNFR1)ectodomain,whichiscontrolledbyTNF- converting enzyme (TACE; also known as ADAM17), a disintegrin metalloproteinase (3, 4). Here, Rowlands et al. provideevidencethatinthelungmicrovascular endothelium, administration of soluble TNF- (sTNF-) increased cytosolic levels of Ca 2+ via inositol- 1,4,5-triphosphatemediated(IP 3-mediated)releasefromtheER(5).Theincrease incytosolicCa2+wasfollowedbyarapid increaseinmitochondrialCa2+,leadingto ariseinmitochondrialROSgeneration fromcomplexIIIoftheelectrontransportchain.TheseROSwererequiredfor
The Journal of Clinical Investigation http://www.jci.org

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Figure 1
Role of mitochondrial Ca2+ and TACE in modulating inflammation of the pulmonary endothelium. (A) During regulated inflammatory response, sTNF- increases mitochondrial Ca2+ and ROS, leading to TACE-mediated TNF receptor ectodomain shedding in endothelial cells. (B) During overwhelming sepsis, increased Ca2+ influx into the mitochondria leads to its dysfunction and to cell death. (C) Restoring the mitochondrial function by decreasing Ca2+ influx into the mitochondria may be a potential therapeutic target during sepsis.

Ca 2+ uniporter (MCU) and rapid mode of uptake channels. This process also involvesthehigh-conductancepermeabilitytransitionpore(PTP),whichspans theouterandinnermitochondrialmembraneandcanallowrapidiontransfer, leading to mitochondrial swelling and dysfunction.ThemolecularCa2+signaling machinery is complex, and studies intotheroleplayedbythemanychannels involvedhasbeenlimitedbyincomplete identificationoftheproteinscomprising them, limiting the development of geneticorspecificpharmacologictools. Instead,investigatorshavereliedonstudiesusingrelativelynonspecificinhibitors, for example, ruthenium red to inhibit MCUandcyclosporineAtoinhibitthe PTP. Interpretation of these studies is complicatedbytheoff-targeteffectsof theseagents.Recently,substantialprogresshasbeenmadeinidentifyingthecriticalproteinconstituentsofthesechannels.Forexample,Perrochietal.recently identified the mitochondrial calcium

uptake1(MICU1)channel,providinga novelmoleculartargettopreventmitochondrialCa2+overload(10). Mitochondrial Ca2+ and ROS signaling in response to TNF Duringoxidativephosphorylation,electronsfromreducedsubstratesaretransferredtoO2throughachainofelectron transporters, and the energy provided by these electron transfer reactions is usedtotransferprotonsacrossthemitochondrialinnermembrane.Theenergy stored in the resulting chemiosmotic gradientisusedtogenerateATP.During electron transfer, some electrons react with molecular O 2 to produce a superoxide anion that is converted to H 2O 2 by mitochondrial or cytosolic SODs. ComplexesIandIIoftheelectrontransportchainreleaseROSexclusivelyinthe mitochondrialmatrix,whereascomplex III generates ROS on both sides of the mitochondrial inner membrane (11). InfluxofCa2+acceleratesthegeneration
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ofreducedsubstratesfromtheTCAcycle andtherateofseveralreactionsinthe electron transport chain, including O 2 consumptionandROSgeneration.Ca2+ also increases the activity of NO synthase,elevatingNOlevels,whichinturn inhibitcytochromeoxidase(complexIV) andmightalsoincreasetheproduction ofROS.Althoughclassicallyconsidered toxicbyproductsofcellularrespiration, morerecentevidencesuggeststhatROS releasedatmoderatelevelsmayactivate cytosolic signaling pathways and transcriptionalprograms,allowingthecellto adapttoenvironmentalstress(12). The increase in mitochondrial Ca 2+ leads to ATP and ROS production, whichcantriggeradditionalincreasesin Ca 2+asaresultoffurtherextracellular Ca 2+ influx (1315). This represents a tipping point in cell survival: low levelsofROSgeneratedatcomplexIIIcan activateadaptivesignalingpathways,but higherlevelsofROSresultintherelease ofcytochromecandcelldeath(12,16).

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Inaseriesofelegantexperiments,Rowlandsetal.transfectedseveralcustomdesignedfluorophoreprobesintomouse lung microvascular endothelium; confocalmicroscopythenshowedthatthe intracellularCa2+increaseoccursintwo steps(5).Theinitialincreasecorresponds with sTNF- activation of the lung microvascularendotheliumandisassociatedwithexpressionoftheleukocyte adhesionreceptorE-selectinandTACEmediatedTNFreceptorshedding.This initial spike is followed by prolonged elevationofCa2+thatpersistsevenwhen TNF-infusionisstoppedandismediatedbythepurinergicreceptorP2Y2(5). TNF receptor shedding as an antiinflammatory adaptation response Ectodomain TNF receptor shedding is animportantmechanismofadaptation and/or protection against inf lammation. The unanchored receptor binds thesolubleTNF,decreasingitsabilityto transduceintracellularsignals,andthus curtailsthecytokinesproinflammatory effects. TNF receptor inhibition is an acceptedtherapeuticmodalityinmany inflammatorydiseases,andtheTNF- inhibitors etanercept (a recombinant solublefusionproteinconsistingofTNF receptor coupled to the Fc portion of IgG),adalimumab(ahumanantiTNF- antibody), and infliximab (a chimeric IgG1 antiTNF- antibody) are widely usedtherapeutics(17).AnimpairedabilitytoshedorendocytoseTNFreceptors canleadtoinflammatorydiseases.For example,amutationinthegeneencoding TNFR1thatimpairsreceptorshedding causesTNFreceptorassociatedperiodic syndrome(TRAPS),anautosomal-dominant autoinf lammatory disease (18). ConsistentwiththefindingsofRowlands etal.(5),aprotectiveroleofTNFR1sheddingbyTACEwasalsoreportedinTNF- sensitizationofhepatocytestoFas-mediateddeath(19). Relevance to sepsis Sepsisremainsacommonclinicalproblem in the United States; therapeutic optionstoaddresstheresultingmultiorganfailurearelimited,andthemortality inpatientswithsepsisremainsunacceptablyhigh.Sepsisoccursinpatientswith overwhelminginfectionsandleadstothe inabilityofcellstousetheO2delivered to them. In patients with septic shock,

cardiacoutputisnormalorevensupranormal,andthedeliveryofO2totissues after acute resuscitation is normal or increased.However,theresponsetoinfectioninpatientswithsepsiscanresultin impairedmitochondrialfunction,which maycontributetocelldeathandmultiorgandysfunction(Figure1Bandrefs.8, 20).Wereasonthatinseveresepsis,there isamassiveinfluxofCa2+,partofwhichis takenupbymitochondriaviatheabovedescribedCa2+channels.Thisrapidinflux ofCa2+leadstoswellingandmitochondrialdysfunction,which,inconjunction with proinflammatory TNF signaling, contributestocelldeath. Rowlands et al. provide evidence that controlled TNF infusion leads to sustainedmitochondrialCa2+elevationand mitochondrial ROS generation in the pulmonaryendothelium(5).Atthelevels they observed, these ROS-activated signalingpathwaysresultedinTNFreceptorsheddingviaTACE,therebylimiting theinflammatoryresponse(Figure1A). Alimitationofthisstudyandofmost researchinthisareaisthatresearchers useamodelofmoderatesepsistoelucidatemechanismsunderlyingtheresponse toTNF-.Itistemptingtospeculatethat higherlevelsofsTNF-inmoreseveresepsiswouldleadtoinfluxofCa2+andthat thesesamepathwayswouldcausemitochondrialdysfunctionandperhapsdeath. AntiTNF-therapyhasbeenineffectiveinthetreatmentofsepsisandcriticalillness,buthasbeenusedwithsuccess in rheumatoid arthritis, Crohn disease, ankylosingspondylitis,andotherchronic inflammatorydiseases.Thistherapyhas been associated with potentially severe side effects, including tuberculosis and seriousbacterialandopportunisticinfections.ThepathwaysRowlandsetal.have identified(5)andtherecentdiscoveryof MICU1protein(10)areencouragingand highlightmechanismsthatmightbetargetedtoamelioratetheinflammationand organ dysfunction observed in patients withsepsis(Figure1C). Acknowledgments TheauthorsaresupportedbyNIHgrants R37-HL48129, HL-85534, and PO1HL71643. Address correspondence to: Jacob I. Sznajder, Pulmonary and Critical Care Medicine, Northwestern University, Feinberg School of Medicine, 240
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E.Huron,McGawM-300,Chicago,Illinois60611,USA.Phone:312.908.7737; Fax: 312.908.4650; E-mail: j-sznajder@ northwestern.edu.

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